The increasing frequency of drought and heatwaves due to global climate change poses a severe threat to desert vegetation. However， the mechanisms by which desert shrubs coordinate hydraulic safety and thermal-tolerance to cope with compound drought-heat stress remain unclear. Five sympatric Tamarix species in the arid region of Northwest China were investigated in this study. By combining 24 h field measurements of gas exchange parameters and leaf water potential with the determinations of physiological traits（xylem embolism vulnerability（P50）， turgor loss point（Ψtlp）， and semi-lethal high temperature（LT50））， we systematically evaluated their adaptive strategies. The results showed that： （1）There was significant interspecific differentiation in drought and heat resistance among the five species. Tamarix hispida exhibited the strongest embolism resistance（P50=-2.20 MPa） and thermal tolerance（LT50=66.5 ℃）， while T. hohenackeri showed the highest gas exchange rates but the weakest resistance. （2）Thermal tolerance（LT50） was significantly negatively correlated with embolism resistance（P50） and significantly positively correlated hydraulic safety margins（HSM）， confirming a high degree of coordinated adaptation rather than a trade-off between hydraulic safety and thermal tolerance in desert plants. （3）Principal Component Analysis（PC1 explained 92.4%） revealed a continuous spectrum of adaptive strategies： ranging from an “Acquisitive-Cooling” strategy represented by T.hohenackeri， to a “Conservative-Tolerance” strategy represented by T.hispida. This study reveals niche differentiation mechanisms based on hydraulic-thermal coupling and highlights the importance of prioritizing species with “Conservative-Tolerance” strategies for vegetation restoration under future hotter and drier climate scenarios.
To clarify stage-specific differences in the functional status of a typical sand-fixing shrub following stubble treatment in an ecologically fragile zone， we investigated approximately 50-year-old Haloxylon ammodendron individuals in the Minqin desert-oasis ecotone， Gansu Province， China. Four treatments were compared： no stubble， half stubble by branch number（50%）， half stubble by height（50%）， and complete stubble. Approximately two years after treatment， architectural traits， water-related functional traits， carbon acquisition traits， non-structural carbohydrate（NSC） contents during the peak growing season， and fine-root segment traits from the 80-90 cm soil layer were measured. Correlation analysis， principal component analysis， and the entropy-weighted TOPSIS method were used to evaluate trait associations and overall functional performance among treatments. The results showed that the multidimensional functional trait configuration of H. ammodendron varied among stubble treatments. Compared with no stubble and complete stubble， the two half-stubble treatments generally showed better aboveground architectural status and functional maintenance characteristics. Half stubble by height（50%） demonstrated relatively better performances in mean crown width， number of living branches， native water transport capacity， hydraulic safety margin， and organ NSC contents. In contrast， half stubble by branch number（50%） showed certain advantages in net photosynthetic rate， fine-root segment length， and specific root length. Correlation and principal component analyses revealed associations among native water transport capacity， hydraulic impairment， stomatal regulation， chlorophyll content， NSC accumulation， and fine-root segment traits， suggesting linked variation among multiple functional traits following stubble treatment. The entropy-weighted TOPSIS evaluation indicated that half stubble by height（50%） had the highest comprehensive score， followed by half stubble by branch number（50%）， whereas no stubble and complete stubble exhibited lower overall functional performance. These results indicated that， for approximately 50-year-old H. ammodendron individuals observed two years post-treatment， half stubble by height（50%） showed relatively better performance in aboveground architecture， maintenance of native water transport capacity， and growing-season non-structural carbohydrate accumulation. This study provides a stage-specific reference for stubble management of aging H. ammodendron plantations in desert-oasis ecotones.
Light is an indispensable environmental factor for plant growth and development. However， UV-B radiation interferes with normal growth processes， causing oxidative damage and inhibiting photosynthesis. To explore the morphological and physiological characteristics and underlying mechanisms of poplars to UV-B stress， dioecious Populus cathayana were used as materials. The cuttings were subjected to UV-B stress alone or combined with exogenous applications of exogenous phytohormones（indole-3-acetic acid（IAA）， 1-aminocyclo-propanecarboxylic acid（ACC）， methyl jasmonate（MeJA）， salicylic acid（SA） and melatonin（MT））. A series of indictors were conducted， including leaf photosynthetic parameters， antioxidant activities， hormone contents， relative expression levels of related genes， anthocyanin contents in stems and leaves， as well as principal component analysis（PCA）. Results showed that under UV-B stress， the net photosynthetic efficiency decreased， and the reactive oxygen species（ROS） molality increased in P.cathayana leaves， with male exhibiting stronger tolerance to UV-B than female P.cathayana. Exogenous hormone applications alleviated UV-B stress-induced damage by improving leaf photosynthetic performance and regulating ROS homeostasis. Specifically， IAA， ACC， and MT treatments exerted more pronounced alleviating effects on female than that on male P.cathayana under UV-B stress. SA application significantly reduced the H2O2 content in leaves. MeJA application promoted organ anthocyanin accumulation， decreased leaf H2O2 content， and enhanced net photosynthetic efficiency and UV-B tolerance on P. cathayana. In addition， the relative expression levels of PtrJAZ1/2/3/5/9 genes were significantly upregulated under both UV-B stress and MeJA treatment， suggesting that these genes may act as key regulators in the JA signaling pathway， mediating anthocyanin synthesis and stress responses. In conclusion， males displayed greater physiological plasticity and stress resistance potential than female P. cathayana under UV-B radiation stress. This study provides practical insights for stress-resistant plant cultivation and hormone regulation in practices.
To investigate the function of PsnSWEET7 in nitrogen response and regulation of carbon and nitrogen metabolism， the transgenic lines overexpressing PsnSWEET7 were generated in Populus simonii × P. nigra via Agrobacterium-mediated transformation. The effects of this gene on plant growth and carbon-nitrogen metabolism were investigated by subcellular localization and phenotype analysis，as well as determinations of physiological indices and gene expression under treatments with different concentrations of ammonium nitrate （NH4NO3）. The PsnSWEET7 protein was localized to the plasma membrane. Under NH4NO3 treatments， compared with the wild type， the plant height and ground diameter of PsnSWEET7 overexpressing plants increased by 17.3% and 17.8%， respectively（P<0.05）； the mass fractions of sucrose， glucose， soluble protein and free amino acids in stems increased by 20.1%， 25.4%， 9.3% and 7.5%， respectively（P<0.05）； the cellulose mass fraction increased by 10.5%（P<0.05）， while the lignin mass fraction decreased by 12.2%（P<0.05）. Under normal and high nitrogen conditions， the activities of vacuolar invertase and cell wall invertase were significantly elevated in transgenic plants. This study indicates that PsnSWEET7 can remodel the carbon and nitrogen metabolism of P.simonii×P. nigra by regulating sugar transport， thereby promoting plant growth， and provides new genetic resources and theoretical basis for the molecular breeding of high-yield and high-quality poplars.
Tonoplast intrinsic proteins（TIPs）， as important members of the aquaporin family， play crucial roles in plant water balance and stress responses. In this study， the function and molecular mechanism of the PagTIP1；3 gene in response to salt stress were investigated in the leaves of wild-type 84K poplar（Populus alba×P.glandulosa） and PagTIP1；3 knockout lines. The results showed that under normal conditions， knockout of the PagTIP1；3 gene led to decreases in chlorophyll content， net photosynthetic rate， and plant height. Under salt stress， the knockout lines exhibited aggravated wilting and yellowing， significantly reduced plant height， and marked decreases in photosynthetic physiological parameters， including maximum photochemical efficiency， electron transport rate， and relative chlorophyll content， compared with the wild type. Transcriptome analysis revealed that the expression patterns of key genes involved in the photosynthetic pathway（including PagLHCB7， PagGAPCP-2， PagPSY， and PagRCA） were significantly altered in the PagTIP1；3 knockout lines in response to salt stress. In summary， it is speculated that PagTIP1；3 deficiency disrupted the transcriptional response of photosynthesis-related genes in poplar under salt stress， thereby impairing photosynthetic electron transport and photoprotection capacity， disturbing growth and photosynthetic physiological homeostasis， and ultimately significantly reducing salt tolerance in poplar.
This study aimed to investigate the molecular function of the C2H2-type zinc finger transcription factor PtrZAT11 in the process of wood formation in poplar. The expression pattern of PtrZAT11 in the xylem at different stem internodes of Populus trichocarpa was analyzed by RT-qPCR. The subcellular localization of the PtrZAT11-GFP fusion protein was observed using a PEG-mediated protoplast transient transformation system. A double mutant of PtrZAT11 and its homologous gene PtrZAT11h was generated using CRISPR-Cas9 technology. Growth parameters and stem anatomical structures of the mutant were subsequently analyzed. The results indicated that PtrZAT11 was significantly highly expressed in stem internodes during the secondary growth stage， and its protein was localized in the nucleus. Compared with the wild type， the ptrzat11 ptrzat11h double mutant exhibited an approximately 31.5% increase in plant height and a significant increase in the number of stem internodes， while no significant differences were observed in ground diameter or internode length. In the 12th internode of the mutant， the width of xylem ring and proportion of xylem cell area were significantly reduced. Additionally， the number of fiber cells per unit area decreased， whereas the number of vessel cells increased. As a nuclear-localized transcription factor， PtrZAT11 participates in wood formation in poplar by influencing the differentiation quantity of xylem cells to regulate xylem tissue organization. It may also coordinate the balance between primary and secondary growth in poplar.
To systematically reveal the synergistic regulatory mechanisms of thinning intensity and water-nitrogen addition on leaf functional traits in plantations， this study conducted a split-plot experiment with Populus tomentosa plantations in the Yellow River Flood Plain. Two thinning intensities（50% and 75%） and three water-nitrogen treatments（water-nitrogen addition， single water addition， and control） were established. The response characteristics and coupling relationships were analyzed from three dimensions： leaf water status， structural construction， and nutrient stoichiometry. The results indicate that： （1）Leaf relative water content remained generally stable， whereas leaf dry mass water content exhibited high sensitivity to thinning and its interaction with water-nitrogen treatments， showing a significant increase of 43.05% under 75% thinning with water-nitrogen addition. （2）Structural traits showed no significant overall changes， with low variation in leaf dry matter content and high variation in specific leaf mass， demonstrating potential plasticity. （3）Among nutrient traits， leaf carbon content remained relatively stable， whereas leaf nitrogen and phosphorus contents as well as their stoichiometric ratios responded significantly to the treatments. The leaf N∶P ratio（mass ratio）ranged from 11.06 to 18.46， with an average value of 13.94， indicating that the P.tomentosa plantations in the study area were generally under nitrogen limitation. （4）Multivariate analysis revealed significant coupling relationships among leaf water content， structural traits， and nutrient allocation， with leaf dry mass water content and specific leaf mass identified as key traits driving nutrient partitioning. （5）Principal component analysis revealed that the first two principal components cumulatively explained 66.4% of the variation. Thinning at 75% promoted leaf adjustment towards high resource-use efficiency， while 50% thinning maintained a conservative strategy. Different water and nitrogen treatments primarily diverged along the nutrient structure dimension， with the single water addition treatment showing strong association with nutrient ratio traits. Overall， thinning and water-nitrogen regulation drive coordinated changes in leaf functional traits by altering resource availability， thereby influencing plant resource utilization strategies.
This study adopted a two-factor （cultivar and waterlogging） completely randomized design to elucidate the responses of poplar trees to waterlogging stress. One-year-old seedlings of five main poplar cultivars-Populus deltoides ‘Xianglin 90’（XL90）， P. deltoides ‘Jianghuai No.1’（JH1）， P. deltoides ‘Wanlin No.1’（WL1）， P. deltoides ‘Zhonghe 1’（2025）， and P. deltoides ‘Nanyang 1’（Juba）-were subjected to three treatments： normal irrigation（CK）， shallow waterlogging（DSY）， and deep waterlogging（GSY）. Growth indices and chlorophyll fluorescence parameters were systematically measured， and the carbon（C）， nitrogen（N）， and phosphorus（P） contents as well as their stoichiometric ratios in leaves， stems， and roots were analyzed. The results showed that waterlogging stress significantly reduced the survival rate of all cultivars and inhibited the growth of tree height and basal diameter. Significant differences in waterlogging tolerance were observed among the cultivars： XL90 exhibited a relatively higher survival rate（67%） and better growth performance under GSY； Juba showed the highest survival rate（83%） under DSY but was sensitive to GSY； JH1， WL1， and 2025 experienced severe growth inhibition under both waterlogging treatments， and their mean growth parameters under DSY might be biased by individual variation due to low survival rates. Chlorophyll fluorescence analysis showed that the GSY treatment significantly decreased the actual photochemical efficiency（ΦPSⅡ） and photochemical quenching coefficient（qP） in all cultivars， whereas qP under DSY did not decrease significantly compared with CK. Waterlogging stress significantly altered the C， N， and P contents in various organs， manifested by increased leaf C content， decreased leaf N content（except for WL1）， and increased N content in stems and roots. Except for JH1， the P content in roots showed an increasing trend. Stoichiometric analysis indicated that for most cultivars， leaf C∶N and N∶P ratios were changed significantly with increasing waterlogging intensity， while the response of C∶P exhibited clearly cultivar-specific difference. Comprehensive evaluation indicated that the waterlogging tolerance of the five poplar cultivars was ranked as XL90>Juba>2025>JH1>WL1. This study elucidated the mechanisms underlying cultivar differences in responding to waterlogging stress from the perspectives of photosynthetic physiology and ecological stoichiometry.
Fluoroquinolone antibiotics（FQs） are widely detected in agricultural irrigation water， livestock wastewater， and coastal wetlands， posing potential stress to plant growth and ecological processes. Taking ofloxacin（OFX） and enrofloxacin（ENR） as representative compounds， this study employed physiological and biochemical assays in combination with transcriptomic analysis to systematically elucidate the physiological responses and molecular regulatory mechanisms of Suaeda salsa under FQs stress. The results showed that both OFX and ENR induced significant oxidative stress in both the aboveground and belowground parts of S. salsa. Comparatively， ENR caused more pronounced lipid peroxidation， which was accompanied by the significant differential expression of genes related to phenylpropanoid metabolism and ABC（ATP-binding cassette） transporters. In contrast， OFX stress primarily impaired photosystem structure and function， while simultaneously enhancing antioxidant defense through the regulation of the WRKY transcription factor family and MAPK（mitogen-activated protein kinase） signaling pathways. Furthermore， both antibiotic treatments promoted the glycolysis-pyruvate metabolism pathway， thereby providing sustained energy supply and reducing power to support stress resistance. Collectively， under FQs stress， S. salsa exhibited a multi-level adaptive response strategy driven by the coordinated regulation of structural defense， metabolic reprogramming， and signaling pathways.
To systematically elucidate the growth response characteristics of pigeon pea（Cajanus cajan） seedlings under different concentrations of salt stress and the dynamic changes in antioxidant enzyme activities and flavonoid metabolite accumulation， uniformly grown pigeon pea seedlings were used as plant materials. The seedlings were treated with 0， 50， 100， 150， and 200 mmol⋅L-1 NaCl solution. Phenotypic observation and root scanning analysis were conducted at 1， 3， 5， and 7 days after treatment. Root morphological parameters， including root length， surface area， projected area， volume， and average diameter， were measured. Meanwhile， the activities of superoxide dismutase（SOD） and peroxidase（POD） were determined. In addition， samples collected under 150 mmol⋅L-1 NaCl treatment at different time points were analyzed using UPLC-MS/MS for qualitative and quantitative profiling of flavonoid metabolites， combined with analysis of related gene expression changes. Results showed that salt stress significantly inhibited the growth of pigeon pea seedlings in a concentration-dependent manner. With increasing salt concentration and prolonged treatment duration， seedlings exhibited inhibited primary root elongation， restricted lateral root development， and reduced shoot growth， with the inhibitory effect becoming more pronounced after 3 days of treatment. Root morphological parameters generally increased over time； however， the magnitude of increase was significantly reduced under 100 mmol⋅L-1 and higher NaCl treatments， whereas 50 mmol⋅L-1 NaCl showed a promoting effect on root growth at certain time points. The average root diameter showed relatively small variation under 50 mmol⋅L-1 NaCl treatment but slight increase under moderate to high salt conditions. SOD activity increased significantly under moderate salt concentration and then stabilized， while POD activity continuously increased with increasing salt concentration and treatment duration. Under 150 mmol⋅L-1 NaCl treatment， flavonoid metabolites in roots exhibited stage-dependent accumulation patterns. Root stilbene acid mass concentration showed an overall increasing trend， isovitexin mass concentration fluctuated markedly， genistin displayed induced accumulation， and genistein mass concentration significantly increased at later stages of treatment. Gene expression analysis showed that the expression levels of CcPAL2， Cc4CL4， and CcCHS3 were downregulated， while CcC4H1 was significantly upregulated， and CcCHI2 and CcIFS1 were also induced. In conclusion， salt stress inhibited the growth of pigeon pea seedlings and showed clear time- and concentration-dependent effects. Antioxidant enzyme activities and flavonoid metabolite levels dynamically changed during the stress process， and the key genes in the flavonoid biosynthesis pathway exhibited differential regulation under salt stress conditions.
Chlorogenic acid（CGA） plays an important role in regulating plant disease resistance. Under pathogen stress， plants typically accumulate various secondary metabolites that are closely associated with stress tolerance. Among these， CGA can enhance plant disease resistance by modulating secondary metabolism； however， the underlying mechanisms remain poorly understood. To further elucidate these mechanisms， Scutellaria baicalensis plants treated with exogenous CGA were subjected to pathogen infection. Leaf chlorophyll content， malondialdehyde（MDA） content， lesion area， and flavonoid content were determined， and transcriptome sequencing coupled with cis-acting element analysis of enzyme-coding genes were performed.The results showed that CGA significantly enhanced the resistance of S. baicalensis to leaf spot disease and influenced the accumulation of the flavonoids naringenin and apigenin. Compared with the control， CGA-treated plants exhibited smaller lesion area， higher chlorophyll level， and lower MDA content. Transcriptome analysis of CGA-treated samples identified 563 differentially expressed genes（DEGs）， which were mainly enriched in pathways including phenylpropanoid biosynthesis， starch and sucrose metabolism， linoleic acid metabolism， galactose metabolism， and flavonoid biosynthesis. Notably， key flavonoid biosynthetic enzyme genes-SbPAL， SbC4H， Sb4CL1， Sb4CL2， SbFNS， SbHCT1， and SbHCT2-were significantly upregulated. Cis-acting element analysis further revealed that the promoter regions of these genes were enriched in binding sites for transcription factors such as bHLH， MYB， and GRF.In conclusion， this study demonstrates that exogenous CGA likely enhances the resistance of S. baicalensis to leaf spot disease by promoting the expression of key genes in the flavonoid biosynthesis pathway， thereby increasing flavonoid accumulation.
To investigate the response patterns to nitrogen forms and determine the optimal nitrogen supply form for enhancing drought tolerance， seedling morphology， biomass， needle water status， and osmoprotectant mass fraction were examined in two-year-old P.koraiensis seedlings under drought levels（moderate and severe drought， with full irrigation as the control） and nitrogen forms（nitrate nitrogen， ammonium nitrogen， and glycinate nitrogen， with no nitrogen application as the control）. The results indicated that the interaction between drought levels and nitrogen forms significantly affected the root morphology， root and needle biomass， seedling quality， needle water status， and osmoprotectants in P.koraiensis seedlings（P<0.05）. Under full irrigation， seedlings fertilized with ammonium nitrogen exhibited significantly superior performances in seedling height， total root length， total root area， root biomass， Dickson’s quality index， and osmoprotectant mass fraction compared to those fertilized with glycinate nitrogen（P<0.05）. Under moderate drought， seedlings fertilized with glycinate nitrogen showed no significant differences in seedling height， root collar diameter， total root volume， root tip number， biomass in various organs and the whole plant， Dickson’s quality index， needle water status， and proline mass fraction compared to those fertilized with ammonium nitrogen. Under severe drought， seedlings fertilized with glycinate nitrogen demonstrated significantly better performances in root collar diameter growth， total root length， total root area， root biomass， needle biomass， total biomass， Dickson’s quality index， needle water status， and needle osmoprotectant mass fraction compared to those fertilized with inorganic nitrogen（P<0.05）. The response of P. koraiensis seedlings to nitrogen forms was significantly affected by drought levels. Under full irrigation， inorganic nitrogen（especially ammonium nitrogen） favored seedling growth and osmoprotectant accumulations. However， under severe drought， glycinate nitrogen improved seedling water status by promoting osmoprotectant accumulations， being more beneficial for seedling growth than inorganic nitrogen. This study elucidates the growth response of P. koraiensis seedlings to different nitrogen forms under drought stress， providing theoretical basis and technical reference for cultivating drought tolerant seedlings through nitrogen application in nurseries.
The basal petal blotch of Paeonia rockii is the primary genetic source of blotch traits in cultivated tree peonies. To clarify the timing of blotch initiation， its expansion pattern， and the relationship between blotch development and pigment accumulation， basal blotch formation at seven developmental stages were systematically examined in ten P.rockii cultivars. At stage S2， single pigmented cells first appeared and gradually developed into pigment cell clusters， consisting of a deeply pigmented central cell surrounded by lightly pigmented peripheral cells. Blotch expansion occurred through the proliferation and enlargement of these pigment cell clusters. The blotch outline was initially established at stage S3， preceding the visible coloration of the petal background， and a second peak of pigment cell proliferation was observed at stages S6-S7. During S2-S3， significant decreases in L* and b* values and an increase in a* value were recorded in the blotch region. Anthocyanins were detectable in the blotch as early as S2， with cyanidin-3，5-di-O-glucoside（Cy3G5G） and peonidin-3，5-di-O-glucoside（Pn3G5G） as the predominant components. Linear regression analysis indicated that Pn3G5G had strong explanatory power for changes in L*， a*， and b* values and was the key anthocyanin contributing to the formation of purple-red blotches. Pelargonidin-3，5-di-O-glucoside（Pg3G5G） may be involved in the formation of black blotches. In summary， blotch formation in P. rockii is a dynamic developmental process characterized structurally by the expansion of pigment cell clusters and biochemically by the spatiotemporally specific accumulation of anthocyanins.
The findings of this study reveal that a species（Petrocosmea condorensis Pellegr.）， described in Petrocosmea one hundred years ago， in fact represents an independent genus in Gesneriaceae. This species is characterized by a flat-faced corolla， suborbicular leaf blades， linear filaments， and conical-ovoid capsules dehiscing into two valves， exhibiting a series of unique morphological characters. Molecular phylogenetic analyses demonstrated that this species belonged to the subtribe Loxocarpinae（Tribe Trichosporeae） within the subfamily Didymocarpoideae of Gesneriaceae and is sister to a clade comprising several other genera. Based on morphological and molecular phylogenetic evidence， we here elevated this species to a new genus， Zhenyuenes， with the establishment of a new combination.
To establish an efficient polyploid induction protocol for Primula filchnerae， seeds and shoot apices were treated with colchicine using the seed immersion method and the cotton ball dropping method. The effects of different colchicine concentrations and treatment durations on germination rate， mortality rate and tetraploid induction rate were compared. Flow cytometry and root-tip chromosome counting were used for ploidy detection and verification. The results showed that colchicine treatment generally suppressed seed germination and increased seedling mortality rate， but it significantly increased the tetraploid induction rate at the optimal concentration. For seed treatment， the highest germination rate（77.67±3.51）% occurred under 0.02 g⋅L-1 colchicine treatment for 24 h， while the lowest mortality rate（2.16±0.93）% and the highest tetraploid induction rate （30.12±4.02）% occurred under 0.05 g⋅L-1 colchicine treatment for 24 h. For the shoot apex treatment， 0.05 g⋅L-1 colchicine treatment had the highest tetraploid induction rate of （40.00±10.00）%. Flow cytometry analysis revealed that the G0/G1 peak of tetraploid plants was significantly shifted from that of diploids， and chromosome counting confirmed that the diploid chromosome number was 2n=2x=24 and the tetraploid chromosome number was 2n=4x=48. Morphological and cytological comparative analysis indicated that the leaf and flower-related traits such as growth potential and ornamental value of tetraploid plants were significantly superior to that of diploid plants. In addition， tetraploids had an increased stomatal size and a decreased stomatal density compared to diploids， which is consistent with typical cytological features of polyploids. In summary， this study established an efficient colchicine-induced polyploidization and ploidy identification protocol for P. filchnerae， and provides technical support for polyploid breeding and germplasm innovation in the species.
Photoperiod serves as the key environmental cue governing seasonal growth and growth cessation in forest trees. Within the perception and transduction of photoperiodic signals， LUX ARRHYTHMO（LUX）， a core component of the circadian clock Evening Complex（EC）， plays a pivotal role. In the present study， a total of 47 BpLUX homologous genes were identified through genome-wide analysis in the reference genome of Betula platyphylla. Bioinformatic analyses revealed that BpLUX1， BpLUX5， and BpLUX18 harbor typical Evening Element（EE） cis-acting elements. All three genes possessed characteristic MYB conserved domains， while BpLUX1 and BpLUX5 additionally exhibited typical circadian rhythmic expression patterns. Using CRISPR/Cas9-mediated genome editing， loss-of-function mutants for BpLUX1（bplux1） were successfully generated， whereas targeted knockout of BpLUX5 proved unsuccessful. Phenotypic characterization demonstrated that under short-day（SD） conditions， wild-type birch ceased growth almost entirely by day 19 in response to shortened photoperiods， whereas bplux1 mutants remained actively growing. Furthermore， while no significant difference in plant height was observed in wild-type plants， bplux1 mutants displayed significantly increased height， exhibiting a delayed growth cessation phenotype. These findings indicate that BpLUX1 is a key gene mediating short-day responses and growth cessation in birch. Transcriptome analysis further revealed that under SD conditions， eleven differentially expressed genes（DEGs） were down-regulated in bplux1 mutants， including BpRCAR1 （Regulatory Component of ABA Receptor 1） involved in ABA （abscisic acid） signaling， whereas seven DEGs were up-regulated， including BpRGL1（RGA-Like1） associated with GA （gibberellin） signaling. Notably， potential binding sites between BpLUX1 and key DEGs in ABA and GA signaling pathways were identified， suggesting that BpLUX1 may influence growth cessation through modulation of phytohormone signaling. Collectively， this study provides a theoretical basis for understanding the role of BpLUX1 in photoperiod-regulated growth cessation in birch.
In the present study， the glutathione S-transferase gene（MiGST） from Meconopsisintegrifolia was cloned， and the biological function through Arabidopsisthaliana transformation was characterized. This study could provide the theoretical basis for flavonoid development and utilization in different tissues of M. integrifolia. The MiGST gene cloned was analyzed by using bioinformatics methods. Then， the tissue expression patterns were examined by real-time fluorescence quantitative polymerase chain reaction（RT-qPCR）. Subsequently， A. thaliana was transformed using the floral dip method， and the total flavonoid content in T1 transgenic lines was determined by the Al（NO3）3 colorimetric assay. The results showed that the coding sequence（CDS） of MiGST was 702 bp in length， which encoded a 234-amino acid protein. The protein molecular weight was 25 861.91 Da with a theoretical isoelectric point of 5.84. Phylogenetic analysis showed that MiGST protein was the most closely related with GST protein from Papaver somniferum. Meanwhile， MiGST gene exhibited the tissue-specific expression， with the highest level in flowers and the lowest in stems. Besides， the total flavonoid contentwithin A. thaliana overexpressing MiGST gene was a 1.27-fold of that of the wild-type one. In conclusion， this study confirmed that overexpression of MiGST gene significantly enhanced the total flavonoid content in transgenic A. thaliana， suggesting that MiGST gene played an important role in the transmembrane transport and accumulation of flavonoids in M. integrifolia.
NAC（NAM， ATAF， and CUC） transcription factors are plant-specific transcriptional regulators that play crucial roles in plant growth， development， and responses to abiotic stresses. To systematically characterize the molecular evolutionary features of theNAC gene family in Catalpa bungei and elucidate its regulatory mechanisms under cold stress， NAC gene family members were comprehensively identified based on the whole-genome sequence of C. bungei. Comprehensive analyses were performed， including physicochemical properties， phylogenetic topology， gene structure conservation， cis-acting elements in promoter regions， cold-responsive expression patterns， and weighted gene co-expression network analysis（WGCNA）. The results demonstrated that a total of 65 CbuNAC genes were identified in the C. bungei genome and classified into four highly conserved subfamilies based on phylogenetic analysis. Chromosomal mapping revealed that these genes were unevenly distributed across 16 chromosomes， displaying a distinct clustered distribution pattern. Gene structure and conserved motif analyses indicated that members within the same subfamily exhibited highly similar intron-exon organization and conserved motif composition，which not only supported the reliability of the phylogenetic classification but also implied potential functional redundancy and evolutionary divergence within subfamilies. Collinearity analysis indicated that segmental duplication， rather than tandem duplication， was the predominant driving force for the expansion and evolutionary divergence of the CbuNAC gene family. Time-series transcriptome analysis under cold stress revealed that CbuNAC15， CbuNAC30， and CbuNAC36 were significantly upregulated， exhibiting strong and temporally dynamic responses to low-temperature stress. Furthermore， WGCNA revealed that the three core genes not only coordinately regulated the classical ICE1-CBF cold-signaling pathway but were also closely associated with downstream genes involved in osmotic adjustment and hormone metabolism. In summary， this study systematically characterized the genomic features of the NAC gene family in C. bungei and identified three key candidate genes associated with cold-tolerance， thereby providing valuable theoretical insights and candidate targets for elucidating abiotic stress adaptation mechanisms in woody plants and advancing marker-assisted breeding.
The stem， as one of the main storage organs of trees， plays a crucial role in wood production； enhancing its storage capacity（sink strength） is of great significance for increasing wood yield. WOX4 belongs to the WUSCHEL-related homeobox（WOX） transcription factor family and functions in maintaining vascular cambium stem cell activity and promoting cell proliferation， thereby regulating secondary growth in stems and roots. It is highly expressed during xylem development in stems and roots. In this study， RT-qPCR was used to analyze the relative expression levels of the PxWOX4a gene in different tissues. In addition， transgenic plants harboring the pPxWOX4a：：GUS construct were subjected to GUS staining to further analyze the expression pattern of the PxWOX4a promoter. The results showed that WOX4a was highly expressed in the stems of Populus×xiaohei. The upstream 1 742 bp promoter region was cloned， and cis-acting element analysis revealed that it contained multiple light-responsive elements， including G-box， I-box， and Box4. A reporter vector（pBI121-pPxWOX4a：：GUS） was generated and transformed into P. alba×P. glandulosa ‘84K’. Molecular analysis confirmed stable integration of the transgene into the genome. RT-qPCR and GUS histochemical staining demonstrated that the PxWOX4a promoter drove strong expression of the GUS gene in stems. These results indicated that the PxWOX4a promoter cloned from Populus × xiaohei exhibited stem-specific and high activity. It therefore provides a useful tool for stem-targeted genetic engineering to regulate wood formation and improve biomass yield in poplar and other forest trees.
This study aimed to optimize the preparation methods of protoplasts from cotyledons and root callus of Andrographis paniculata， and to investigate the key factors affecting their transient transformation， thereby laying a technical foundation for subsequent genetic transformation and gene function research in A. paniculata. The effects of different mannitol concentrations， enzyme concentrations， and enzymatic hydrolysis time on protoplast yield and viability were studied using the enzymatic hydrolysis method. The PEG-mediated transformation method was employed to examine the influence of factors such as PEG concentration and transformation time on transient transformation efficiency， and a transient transformation system for A. paniculata root callus protoplasts was established. The optimal conditions for protoplast preparation from A. paniculata cotyledons were as follows： 0.2 g cotyledons were enzymatically hydrolyzed in a solution containing 4.0% cellulase R-10， 1.5% macerozyme R-10， and 0.6 mol⋅L-1 mannitol at 26 ℃ and 55 r⋅min-1 in darkness for 4 h. Under these conditions， the protoplast yield reached 3.32×10⁵ cells⋅mL-1， with a viability of 90.73%. For root callus protoplasts， the optimal conditions were： 0.4 g 15‑day‑old callus was enzymatically hydrolyzed in a solution containing 4.0% cellulase R-10， 1.0% macerozyme R-10， and 0.8 mol⋅L-1 mannitol under the same conditions for 9 h， yielding 2.18×106 cells⋅mL-1 with a viability of 94.19%. In PEG-mediated transient transformation， the optimal conditions were determined to be 40% PEG 4000 treatment for 30 min， 10 μg of plasmid DNA， and 0.6 mol⋅L-1 mannitol. Under these conditions， the transformation efficiency reached 58.03%. Significant differences existed in the optimal preparation conditions for protoplasts derived from different tissues of A. paniculata， emphasizing the need for tissue-specific optimization. This study established an efficient protoplast preparation and transient transformation system forboth cotyledons and root callus of A. paniculata， providing a reliable technical platform for future gene function analysis and cell biology research in this medicinal plant.
